ATP-independent Activation of Natriuretic Peptide Receptors

Antos, Laura K.; Abbey-Hosch, Sarah E.; Flora, Darcy R.; Potter, L. R.

doi:10.1074/jbc.m505648200

Cited by 39 publications

(33 citation statements)

References 41 publications

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“…ATP-mediated effects are markedly different between GC-A and GC-C in that the presence of ATP increases ANPstimulated activity of GC-A manyfold, in contrast to the more modest increase seen with GC-C and ST (40). We observed no change in the activity of wild type GC-A or mutant receptors in the presence of detergent and ATP (data not shown).…”

Section: Figure 6 Characterization Of Gc-a Linker Mutantsmentioning

confidence: 42%

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module

Saha

Biswas

Kondapalli

et al. 2009

Journal of Biological Chemistry

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Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of ϳ70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand-and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain.In transmembrane receptors a series of conformational changes are required to transmit the information of ligand binding (an extracellular signal) to the interior of the cell, resulting in either altered interaction with signaling intermediates or in the regulation of a catalytic activity present in the receptor. In these multidomain receptors, where the ligand binding and effector domains are present in the same polypeptide chain, the relay of conformational changes is under the exquisite control of post-translational modifications or precise structural alterations.Receptor guanylyl cyclases (GCs) 4 have an N-terminal extracellular ligand binding domain, a single transmembrane domain, and a C-terminal intracellular domain (1). Binding of ligands to the extracellular domain elicits a conformational change that increases the guanylyl cyclase activity of the receptor, resulting in increased cGMP production. The intracellular domain of receptor GCs contains a region that shares considerable sequence similarity to protein kinases and is referred to as the kinase homology domain (KHD). Binding of ATP to the KHD induces a conformational change that regulates cGMP production by the guanylyl cyclase domain (2). Thus, receptor GCs exemplify the intricate interactions between domains in transducing the signal from an extracellular ligand to the interior of the cell.The amino acid sequences of ...

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Section: Figure 6 Characterization Of Gc-a Linker Mutantsmentioning

confidence: 42%

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module

Saha

Biswas

Kondapalli

et al. 2009

Journal of Biological Chemistry

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“…Crude membranes were prepared as described (21). Guanylyl cyclase assays were performed at 37°C for 5 min in a buffer containing 25 mM Hepes pH 7.4, 50 mM NaCl, 0.1% BSA, 0.5 mM 1-methyl-3-isobutylxanthine, 1 mM GTP, 0.5 µM microcystin, 1 mM EDTA, and 1–2 µCi of [α- 32 P]GTP, with either 5 mM MgCl 2 , 1 mM ATP, and 10 nM or 1 µM natriuretic peptide or 1% Triton X-100 with 5mM MnCl 2 being substituted for the MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

Mass Spectrometric Identification of Phosphorylation Sites in Guanylyl Cyclase A and B

Yoder¹,

Stone

Griffin

et al. 2010

Biochemistry

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Guanylyl cyclase A and B (GC-A and GC-B) are transmembrane guanylyl cyclase receptors that mediate the physiologic effects of natriuretic peptides. Some sites of phosphorylation are known for rat GC-A and GC-B, but no phosphorylation site information is available for the human homologs. Here, we used mass spectrometry to identify phosphorylation sites in GC-A and GC-B from both species. Tryptic digests of receptors purified from HEK293 cells were separated and analyzed by nLC-MS-MS. Seven sites of phosphorylation were identified in rat GC-A (S497, T500, S502, S506, S510, T513, and S487) and all of these sites except S510 and T513 were observed in human GC-A. Six phosphorylation sites were identified in rat GC-B (S513, T516, S518, S523, S526, and T529) and all six sites were also identified in human GC-B. Five sites are identical between GC-A and GC-B. S487 in GC-A and T529 in GC-B are novel, uncharacterized sites. Substitution of alanine for S487 did not affect initial ligand-dependent GC-A activity, but a glutamate substitution reduced activity 20%. Similar levels of ANP-dependent desensitization were observed for the wild type, S487A and S487E forms of GC-A. Substitution of glutamate or alanine for T529 increased or decreased ligand-dependent cyclase activity of GC-B, respectively, and T529E increased cyclase activity in a GC-B mutant containing glutamates for all five previously identified sites as well. In conclusion, we identified and characterized new phosphorylation sites in GC-A and GC-B and provide the first evidence of phosphorylation sites within human guanylyl cyclases.

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“…Figure 7 was performed as described for Figs. 1 and 2 except that 300 Ci of [␣-32 P]GTP was added to cold GTP, and conversion of [␣- 32 P]GTP to [ 32 P]cGMP was measured as previously described (3).…”

Section: Methodsmentioning

confidence: 99%

“…In the early 1990s, it was reported that adenine nucleotides are absolutely required for natriuretic peptide-dependent activation of NPR-A and NPR-B (10,12). These data then led to a two-step activation model, which was left unchallenged until 2005, when we reported that ATP is not essential for activation of natriuretic peptide receptors in human embryonic kidney 293 cells highly overexpressing NPR-A or NPR-B (3,35). Because ATP increased the guanylyl cyclase activity of these receptors only at longer but not shorter times, we concluded that ATP stabilizes these receptors but is not required for their activation.…”

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confidence: 98%

Adenine nucleotides decrease the apparentK_mof endogenous natriuretic peptide receptors for GTP

Antos

Potter²

2007

American Journal of Physiology-Endocrinology and Metabolism

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Antos LK, Potter LR. Adenine nucleotides decrease the apparent K m of endogenous natriuretic peptide receptors for GTP. Am J Physiol Endocrinol Metab 293: E1756-E1763, 2007. First published September 11, 2007 doi:10.1152/ajpendo.00321.2007.-Natriuretic peptide receptors A (NPR-A) and B (NPR-B) mediate most effects of natriuretic peptides by synthesizing cGMP. ATP increases the activity of these receptors by an unknown mechanism. We recently reported that a nonhydrolyzable form of ATP, adenylyl imidodiphosphate (AMPPNP), stabilizes but is not required for the activation of NPR-A and NPR-B in membranes from highly overexpressing cells. Here, we repeated these studies on receptors expressed in endogenous settings. Kinetic analysis indicated that both AMPPNP and ATP dramatically decrease the apparent Km of both receptors for GTP but had little effect on the Vmax. The EC50 for AMPPNP decreased as substrate concentration increased whereas the magnitude of the effect was greater at lower GTP concentrations. ATP increased the activity of a mutant receptor containing glutamates substituted for all known phosphorylation sites similarly to the wildtype receptor, consistent with a phosphorylation independent mechanism. Finally, the putative ATP binding sites were investigated. Mutation of the ATP modulatory domain region had no effect, but mutation of K535A dramatically diminished ANP-dependent cyclase activity in a manner that was unresponsive to ATP. Mutation of the highly conserved 630-KSS to AAA (all alanines) resulted in an expressed receptor that had no detectable guanylyl cyclase activity. We conclude that ATP is not required for the initial activation of NPRs but does increase activity over time by reducing the apparent

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ATP-independent Activation of Natriuretic Peptide Receptors

Cited by 39 publications

References 41 publications

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module

Mass Spectrometric Identification of Phosphorylation Sites in Guanylyl Cyclase A and B

Adenine nucleotides decrease the apparentK_mof endogenous natriuretic peptide receptors for GTP

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ATP-independent Activation of Natriuretic Peptide Receptors

Cited by 39 publications

References 41 publications

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module

Mass Spectrometric Identification of Phosphorylation Sites in Guanylyl Cyclase A and B

Adenine nucleotides decrease the apparentKmof endogenous natriuretic peptide receptors for GTP

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Adenine nucleotides decrease the apparentK_mof endogenous natriuretic peptide receptors for GTP